Staining and microscopy protocol for 3D organotypic cultures
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Before starting

This protocol involves the use of the Leica SP5FLIM inverted microscope at PennVet, so you should receive training from Gordon Ruthel at the PennVet Microscopy Core before using this instrument. Microscopic images will be acquired using the Leica Application Suite Advanced Fluorescence (LAS AF) software.

Materials and supplies

antibodies and stains

  • Fluorescein labeled Ulex Europaeus Agglutinin I (UEA I) to detect mucin (Vector labs cat# FL-1061)
  • Rabbit anti-human zona occludens-1 (ZO-1; mAb clone D6L1E), a tight junction protein (Cell Signaling Technology cat# 13663S)
  • Goat anti-rabbit Alexa Fluor®568 to detect ZO-1 (Invitrogen cat# A-11031)
  • Mouse anti-human Ezrin (clone 4A5); a brush border marker (Millipore/Sigma cat# MAB3822-C)
  • Goat anti-mouse Alexa Fluor®700 to detect Ezrin (Invitrogen cat# A-21036)
  • VECTASHIELD® Vibrance™ Antifade Mounting Medium with DAPI (Vector Labs, Cat# H-1800-10).
  • Alexa Fluor® 680 conjugated Phalloidin; stains actin (it is a toxin that bind competitively to the same sites in F actin) - (Invitrogen cat# A22286).

other consumables

  • 4% Paraformaldehyde (formaldehyde) aqueous solution (Electron Microscopy Sciences cat# 157-4-100)
  • Aqueous solution of 10% Triton X-100 (Sigma, USA)
  • Phosphate buffered saline – PBS (ice-cold for rinsing steps)
  • Cell Imaging Dish, 170 μm thich, 35x10 mm (Eppendorf cat# 0030740017)

Fixing and permeabilizing cells

  • For details on recovering beads from STLV culture, see protocol here
  • harvest a 100μl aliquot of the the 3D beads Caco-2 cell culture using a CUT PIPPETE TIP and place it in an eppendorf tube
  • Gently remove media with a pipette tip and wash beads 3x with PBS.
  • Fix cells by removing last PBS wash and adding 200μl of 4% paraformaldehyde. Incubate at room temperature for 10 min.
  • Wash cells three times with ice-cold PBS. Incubate 5 min after each wash.
  • Permeabilize cells by removing last PBS wash and adding 200μl of 0.25% Triton X-100.
  • Incubate at room temperature for 10 min.
  • Wash again 3x.
  • The fixed and permeabilized bead preparation can be used immediately for staining or can be kept at 4 deg for use later.

Immunofluorescent staining

  • Remove PBS from fixed/permeabilized cells
  • Prepare ZO-1 (1/400) and Ezrin (1/400) antibodies by adding 2ul of each to 800ul of PBS. Scale up if more than 800ul is needed.
  • Add enough antibody mix to cover beads
  • Incubate for 1h at room temperature with occasional gentle mixing by inverting.
  • Remove antibody and wash 3x with PBS (5 min each wash)
  • Prepare UEA-FITC (1/400), Alexa Fluor®568 (1/500), and Alexa Fluor®700 (1/500) by adding 2.5ul, 2ul, and 2ul, respectively, in 1ml of PBS.
  • Note If using phalloidin, dilute 1/50 for staining.
  • Add enough antibody mix to cover cells.
  • Incubate for 1h at room temperature with occasional gentle mixing by inverting.
  • Remove antibody and wash 3x with PBS (5 min each wash).
  • Cells are now ready to be mounted and imaged

Preparing for confocal microscopy

  • Using a truncated pipette tip, carefully transfer 1-2 drops of the stained beads to a Cell Imaging Dish and add 1-2 drops of the Vectashield® antifade mounting medium and take it for microscopic observation. Protect from light (cover with alumnium foil) and heat (keep it on ice).

2D cultures

  • If fixing/staining of 2D cultures is also needed, cells should be grown on Cell Imaging Dishes d and fixed/stained in the same manner as 3D cultures above.
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